All living cells can sense their environment. The term directional sensing refers to the ability of a cell to determine the direction and proximity of an extracellular stimulus. Directional sensing is needed to detect morphogens that control differentiation and attractants that direct cell migration, as in chemotaxis. This fascinating response is critical in embryogenesis, angiogenesis, neuronal patterning, wound healing, and immunity. Chemotaxis is strikingly exhibited during the life cycle of the social amoebae, Dictyostelium discoideum. During growth, these cells track down and phagocytose bacteria. When starved, they move towards secreted adenosine 3'-5' cyclic monophosphate (cAMP) signals, form aggregates, and differentiate into spore and stalk cells. The fundamental role of chemotaxis in this simple eukaryote provides a powerful system for its analysis at both genetic and biochemical levels. Both amoebae and mammalian leukocytes use G protein-linked signaling pathways to respond to chemoattractants. Binding of the attractants to receptors of the seven transmembrane helix class leads to the dissociation of the G proteins into alpha and beta/gamma-subunits. In both leukocytes and amoebae, chemoattractants elicit a variety of rapid responses including transient increases in Ca2+ influx, in the intracellular messengers IP3, cAMP and guanosine 3'-5' cyclic monophosphate (cGMP), and in the phosphorylation of myosins I and II. Chemoattractants also induce actin polymerization, most likely through the activation of the Rho family of small guanosine trisphosphatases. All these events rapidly subside in the presence of persistent stimulation. This rapid inhibition may allow a migrating cell to "subtract" the ambient concentration of attractant and more accurately sense the direction of a gradient. Our research program is focused on learning how specific G protein-coupled signaling events translate into complex cellular responses such as cell migration and differentiation. In D.discoideum, genetic analyses have established that the activation of adenylyl cyclase (ACA) requires, in addition to the beta/gamma-subunits of G proteins, a novel protein called CRAC (cytosolic regulator of adenylyl cyclase). We have shown that CRAC, which contains a pleckstrin homology (PH) domain at its N-terminus, translocates rapidly and transiently to the plasma membrane upon addition of a chemoattractant. Remarkably, this translocation occurs selectively at the leading edge of chemotaxing cells. In both leukocytes and amoebae, other PH domain-containing proteins such as PKB (Akt) behave similarly. We have proposed that the appearance of specific binding sites for PH domain-containing proteins at the leading edge of chemotaxing cells spatially targets signaling events. My group is interested in identifying the molecular mechanisms that regulate these pathways and in particular the role of adenylyl cyclase in chemotaxis. Our research plan is comprised of three interconnected specific aims: (1) Identify the molecular mechanisms involved in the activation of CRAC, (2) Study the spatial and temporal localization of adenylyl cyclases in chemotaxing cells, and (3) Identify the mechanisms of activation of adenylyl cyclases in neutrophils. These studies have direct bearing on our understanding of clinically important processes such leukocyte migration to sites of inflammation as well as cancer metastasis.